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Abstract:

Nitro oleic acid and related metabolites are agonists of PPAR-γ.
Surprisingly, nitro oleic acid is a more potent agonist of PPAR-γ,
relative to nitro linoleic acid. Thus, nitro oleic acid and its
metabolites, as well as their pharmaceutically acceptable salts and
prodrug forms, are candidate therapeutics for the treatment of type-2
diabetes, which results from insulin resistance accompanying the improper
functioning of PPAR-γ.

Claims:

1. A pharmaceutical composition comprising (A) an active agent selected
from nitro oleic acid and a metabolite of nitro oleic acid, or a
pharmaceutically acceptable salt or prodrug of said active agent, and (B)
a pharmaceutically acceptable carrier.

6. use of an active agent selected from nitro oleic acid and a metabolite
of nitro oleic acid, or a pharmaceutically acceptable salt or prodrug of
said active agent, for the preparation of a pharmaceutical composition
for treating type-2 diabetes in a subject.

7. A method for gauging efficacy of a treatment for type-2 diabetes,
comprising: (A) obtaining a first and a second sample from a subject
suffering from type-2 diabetes, wherein the samples are obtained at
different times during said treatment; (B) determining blood glucose
levels in said first and second samples; and (C) comparing the blood
glucose level between said samples, whereby a lower blood glucose level
in said second sample is an indicator of efficacy of said treatment.

Description:

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims priority from U.S. provisional application
No. 60/953,360, filed Aug. 1, 2007, which is incorporated here by
reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the use of nitrated fatty acids as
therapeutics for treating type-2 diabetes. Fatty acids are both
physiological energy sources and mediators of signaling events involved,
for example, in inflammation and in energy homeostasis.

[0005] The signaling ability of nitro fatty acids stems predominantly from
their ability to form reversible covalent adducts with nucleophilic
centers of cellular proteins that are implicated in various
transcriptional and cellular signaling processes. In particular,
regulation of signaling activity most often occurs via the covalent
modification of an active site thiol group of a protein target.

[0007] While both nitro oleic acid and nitro linoleic acid interact with
PPAR-γ, little is known about the structural and biochemical
determinants that account for their PPAR-γ activity and the related
downstream activation of gene transcription. Consequently, no systematic
approach exits for the design of pharmacophores that can modulate
PPAR-γ activity.

SUMMARY OF THE INVENTION

[0008] Accordingly, the present invention provides, in one of its aspects,
a pharmaceutical composition comprising (A) an active agent selected from
nitro oleic acid and a metabolite of nitro oleic acid, or a
pharmaceutically acceptable salt or prodrug of such active agent, and (B)
a pharmaceutically acceptable carrier. In a preferred embodiment, the
active agent is nitro oleic acid.

[0009] In accordance with another of its aspects, the invention provides a
method for treating type-2 diabetes, comprising (A) administering to a
subject in need thereof a pharmaceutical composition as described above
and then (B) repeating step (A) at least once. Preferably, the method
further comprises, after at least one repetition of step (A); the
monitoring of the subject for a change relating to type-2 diabetes.

[0010] Pursuant to yet another aspect of the invention, a method is
provided for gauging efficacy of a treatment for type-2 diabetes. This
method comprises (A) obtaining a first and a second sample from a subject
suffering from type-2 diabetes, which samples are obtained at different
times during said treatment; (B) determining blood glucose levels in the
first and second samples; and (C) comparing the blood glucose level
between the samples. In accordance with these steps, a lower blood
glucose level in the second sample is an indicator of efficacy of the
treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a graph depicting serum levels of nitro oleic acid and as
its physiological metabolites as a function of time.

[0012] FIG. 2 is a graph that correlates blood glucose levels in wild-type
(WT) mice on different days after injection with oleic acid, nitro oleic
acid, rosiglitazone, and a vehicle.

[0013] FIG. 3 is a graph that correlates blood glucose levels in
leptin-deficient diabetic ob/ob mice on different days after being
injected with oleic acid, nitro oleic acid, rosiglitazone, and a vehicle.

[0014] FIG. 4 is a graph that correlates the body weights of WT and ob/ob
mice, undergoing treatment with nitro oleic acid, to the body weight of
WT and ob/ob mice that receive oleic acid, rosiglitazone, and a vehicle,
respectively.

[0015] FIG. 5 is a graph that shows the change in insulin sensitivity for
WT mice injected with oleic acid, nitro oleic acid, rosiglitazone, and a
vehicle, respectively

[0016] FIG. 6 is a graph that documents a change in insulin sensitivity of
ob/ob mice, observed after injection with oleic acid, nitro oleic acid,
rosiglitazone, and a vehicle, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Nitro oleic acid and related metabolic products ("metabolites") are
agonists of PPAR-γ. The inventor's discovery that nitro oleic acid
is surprisingly more potent as an agonist of PPAR-γ, relative to
nitro linoleic acid, underscores the prospect, in accordance with the
present invention, of using nitro oleic acid and its metabolites, as well
as their pharmaceutically acceptable salts and prodrug forms, as active
agents in the treatment of type-2 diabetes, which results from insulin
resistance accompanying the improper functioning of PPAR-γ.

[0018] The structural determinants responsible for the potency of nitro
oleic acid and its metabolites at PPAR-γ were illuminated using a
computational model for receptor-ligand interaction. Modeling data
indicate that both arginine-288 (Arg288) and cysteine-285 (Cys285),
present in the ligand binding pocket of PPAR-γ, are important for
binding. For example, the receptor-ligand model indicates an
electrostatic interaction between arginine-288 and the anionic nitro
group of nitro oleic acid, while Cys285 is found to be in a suitable
position for interacting with the olefinic double bond. These
observations indicate that nitro oleic acid activates PPAR-γ via
the covalent modification of its active site thiol, and compounds that
preserve such interactions will activate the receptor in a similar
manner, thus qualifying as a candidate therapeutic for treating type-2
diabetes.

[0019] Analysis of mouse plasma after intravenous administration of nitro
oleic acid illuminates the physiological fate of nitro oleic acid. Nitro
oleic acid is converted in-vivo, to its saturated analog, or can undergo
β-oxidative cleavage to give several short chain products, such as
the corresponding saturated or unsaturated C-10 to C-16 nitrated analogs.
FIG. 1 shows the plasma levels of nitro oleic acid or its metabolic
products as a function of time. For nitro oleic acid as well as its
saturated 18:0 nitrated analog, the curve is biphasic, with peak
concentrations occurring at around 5 minutes following the administration
of nitro oleic acid. In contrast, the plasma levels for the
β-oxidation products are highest at around 60 minutes.

[0020] The presence of β-oxidation products in blood plasma has
important physiological implications. It is believed that the short-chain
metabolites are less hydrophobic than the parent acid. Nevertheless,
these compounds preserve the molecular determinants that are believed to
be important for binding. Additionally, the smaller size the C-10 to C-16
metabolic products will allow these metabolites to partition differently
between the hydrophobic and hydrophilic compartments physiologically.
Such differences in partitioning ratios alter the anatomic distribution,
chemical reactivity, and pharmacological profiles of these metabolites,
by altering their availability to cellular targets. Pursuant to the
invention, the C-10 to C-16 metabolites are also suitable candidate
therapeutics for the treatment of type-2 diabetes, a condition associated
with PPAR-γ dysfunction. See Freeman et al., Chem. Res. Toxicol.
12: 83-92 (1999).

[0021] Supportive of this anti-diabetic indication, nitro oleic acid was
found to improve insulin sensitivity and lower blood glucose levels in
ob/ob mice. In particular, in-vivo results indicate that nitro oleic acid
reduces blood glucose levels without the side-effects of weight gain and
fluid retention associated with Rosiglitazone, a known PPAR-γ
agonist.

[0022] As shown in FIG. 2, nitro oleic acid but not oleic acid maintains a
steady blood glucose level in fed WT mice. The in-vivo results indicate
that nitro oleic acid was at least as effective as Rosiglitazone in
maintaining blood glucose levels. Similar results are observed in
experiments involving ob/ob mice. As seen from the graph in FIG. 3, both
nitro oleic acid and Rosilitazone are effective in reducing blood glucose
levels. However, for mice receiving oleic acid the blood glucose levels
increased over the course of the study. These results, therefore, provide
support for nitro oleic acid's role in lowering blood glucose and as a
candidate therapeutic for the treatment of diabetes.

[0023] In addition to reducing blood glucose levels, no increase in body
weight is observed when nitro oleic acid is administered to mice. As
shown in FIG. 4, the body weights of WT mice receiving nitro oleic acid,
or Rosiglitazone do not change over the course of the study (25 days),
however, the results are substantially different for ob/ob mice. In this
case, the body weight initially decreases for animals receiving nitro
oleic acid (days 0-10) and then remains constant over the latter half of
the study. In contrast, animals receiving Rosilitazone or oleic acid show
a steady increase in body weights over the entire course of the study.

[0024] Without endorsement of any particular theory, the absence of weight
gain in ob/ob mice is believed to occur because physiologically nitro
oleic acid is adduced to plasma, which serves as a "storage system" and
temporarily inactivates the nitrated fatty acid, until it is required for
facilitating a particular signal transduction event. Since activation of
PPAR-γ occurs upon binding free nitro oleic acid, the sequestration
of this molecule prevents the aberrant activation of PPAR-γ or the
transcription of genes that are regulated by this nuclear receptor.

[0025] Further evidence, that nitro oleic acid modulates PPAR-γ
activity via a binding interaction different from that of Rosiglitazone
is provided by the discovery that nitro oleic acid and not Rosiglitazone
improves insulin sensitivity in ob/ob mice. In this regard, for WT mice
receiving either nitro oleic acid or Rosiglitazone, the administration of
insulin results in an initial drop in blood glucose levels followed by an
elevation to normal levels shortly after the administration of insulin as
shown in FIG. 5. In contrast, as seen by the graph in FIG. 6,
administration of nitro oleic acid to ob/ob mice followed by the
administration of insulin causes a substantial decrease in blood glucose
levels. On the other hand, the blood glucose levels in ob/ob mice
receiving Rosiglitazone are unchanged upon administration of insulin.
These results indicate that administration of nitro oleic prior to
insulin enhances insulin sensitivity in ob/ob diabetic mice, while
Rosiglitazone fails to do so. The fact that both nitro oleic acid and
Rosiglitazone exert their blood glucose lowering effect through the
activation of PPAR-γ, indicates that they interact differently with
the receptor and consequently the transcription of genes that regulate
metabolic events that lead to weight gain and fluid retention.

[0026] The importance of nitro fatty acids as signaling molecules has
prompted the development of various procedures for synthesizing these
compounds. For example, Brandchaud et al., Org. Lett. 8: 3931-34 (2006),
and King et al., Org. Lett. 8: 2305-08 (2006), disclose syntheses that
could be used in the context of the present invention. Another suitable
synthesis approach, disclosed in U.S. Patent Publication No.
2007/0232579, involves the direct nitration of an appropriate unsaturated
fatty acid. Accordingly, (Z)-octadec-9-enoic acid (oleic acid) is reacted
with NaNO2 in the presence of phenylselenium bromide and mercuric
chloride under anhydrous conditions to give 9-nitro or 10-nitro oleic
acid. A similar synthetic strategy is believed to nitrate the appropriate
C-10 to C-16 unsaturated fatty acids to give the corresponding nitro
oleic acid metabolic products.

[0027] Thus obtained, the synthetic regioisomers of nitro oleic acid or
their respective C-10 to C-16 metabolites are typically purified prior to
biological use. In one aspect of the invention, therefore, large-scale
purification of the individual isomers is carried out using preparative
high performance liquid chromatography (HPLC), as described in U.S.
Patent Publication No. 2007/0232579. The purified compounds thus obtained
are appropriately formulated prior to in vivo administration.

[0028] A pharmaceutical composition of the invention can include one or
more therapeutic agents in addition to nitro oleic acid or a related
compound, as described above. Illustrative of such therapeutics are
cytokines, chemokines, and/or regulators of growth factors. Additionally,
the invention contemplates a formulation that contains either a single
regioisomer or both regioisomers of nitro oleic acid.

[0029] The pharmaceutical composition can have more than one
physiologically acceptable carrier, too, such as a mixture of two or more
carriers. The composition also can include thickeners, diluents,
solvents, buffers, preservatives, surface active agents, excipients, and
the like.

[0030] The pharmaceutical carrier used to formulate the nitro oleic acid
of the invention will depend on the route of administration.
Administration may be topical (including opthamalic, vaginal, rectal, or
intranasal), oral, by inhalation, or parenterally, for example by
intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection.

[0031] Thus, nitro oleic acid or its metabolites can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, transdermally, intratracheally, extracorporeally, or
topically (e.g., topical intranasal administration or administration by
inhalant). In this regard, the phrase "topical intranasal administration"
connotes delivery of the compositions into the nose and nasal passages
through one or both of the nares and can comprise delivery by a spraying
mechanism or droplet mechanism, or through aerosolization of the nucleic
acid or vector. The latter can be effective when a large number of
subjects are to be treated simultaneously, where "subject" can denote a
human or an non-human animal. Administration of the compositions by
inhalant can be through the nose or mouth via delivery by a spray or
droplet mechanism. Delivery also can be directed to any area of the
respiratory system, such as the lungs, via intubation.

[0032] A pharmaceutical composition of the nitro oleic acid and its
metabolites for parenteral administration, according to the invention,
can include excipients and carriers that stabilize the nitro fatty acid
mimetic. Illustrative of such a carrier are non-aqueous solvents, such as
propylene glycol, polyethylene glycol, vegetable oils, and injectable
organic esters such as ethyl oleate. Additionally, formulations for
parenteral administration include liquid solutions, suspensions, or solid
forms suitable for solution or suspension in liquid prior to injection,
or emulsions.

[0033] Intravenous compositions can include agents to maintain the
osmomolarity of the formulation. Examples of such agents include sodium
chloride solution, Ringer's dextrose, dextrose, lactated Ringer's
solution, fluid and nutrient replenishers, and the like. Also included in
intravenous formulations are one or more additional ingredients that
prevent microbial infection or inflammation, as well as anesthetics.

[0034] Alternatively, pharmaceutical compositions of the salt form of
nitro oleic acid or its metabolites is administered. Illustrative of such
salts are those formed by reaction of the carboxyl group with an
inorganic base such as sodium hydroxide, ammonium hydroxide, or potassium
hydroxide. Additionally, the salt is formed by reacting the carboxyl
group with organic bases such as mono-, di-, trialkyl and aryl amines and
substituted ethanolamines.

[0035] To address concerns that at physiological pH, nitro oleic acid
typically will be a negatively charged molecule, which may have
non-optimal bioavailability and cell-transport kinetics, one may provide
a compound of the invention formulated as a prodrug. Illustrative of such
a prodrug is a pharmaceutically acceptable ester, such as a methyl or an
ethyl ester. The ester acts as a prodrug because non-specific
intracellular esterase convert it to the active form responsible for
eliciting therapeutic effect.

[0036] Type-2 diabetes is a chronic condition that results from a loss of
sensitivity to insulin. As described above, a pharmaceutical composition
of the invention improves insulin sensitivity and, hence, can serve as a
therapeutic for treating type-2 diabetes. Successful treatment of type-2
diabetes typically entails as well an ongoing monitoring of the subject
for changes related to the diabetic condition, e.g., monitoring
physiological levels of different metabolic parameters associated with
this condition. Thus, the subject's blood and urine glucose levels can be
measured to assess how frequently to administer the inventive
composition. Additional markers such as a gain in body weight, frequency
of urination and the levels of glucagon in the blood can be used to
monitor and possibly to modify treatment to best suit the given subject.

[0037] In support of such an anti-diabetic regimen, the present invention
also provides for using one of the above-mentioned active agents to
prepare a pharmaceutical composition for treating type-2 diabetes in a
subject. To this end, different formulation approaches have been
described above.

[0038] In a related vein, the invention encompasses a method for gauging
the therapeutic efficacy of the composition as described above. This
method involves obtaining at least two blood samples from a subject at
different times during treatment and measuring the level of blood glucose
in each sample. Indicative of therapeutic efficacy is a lower level of
blood glucose in the sample obtained at a later time point during
treatment. As shown in FIG. 3, blood glucose levels in ob/ob mice
receiving nitro oleic acid are significantly lower on day 21 than at the
beginning of the study, indicating the therapeutic benefit of nitro oleic
acid in treatment of type 2 diabetes.

Patent applications by Bruce A. Freeman, Pittsburgh, PA US

Patent applications by University of Pittsburgh - Of the Commonwealth System of Higher Education